The precise molecular and genetic mechanisms leading to the complete spectrum of dental and craniofacial disorders in humans remain unknown. Rather than experiencing aberrations of the craniofacial skeleton in isolation, affected individuals frequently manifest co-occurring deficits. The role of deleterious, pleiotropic loci in causing these co-morbid features remains largely unexplored. To approach this problem, we seek to develop a natural model system, Astyanax mexicanus, for powerful genomic analyses. This model system consists of epigean (surface) and subterranean (cave-dwelling) forms that demonstrate numerous degenerative phenotypes. These include craniofacial bone phenotypes (dermal bone fragmentation) and dental aberrations (an ectopic maxillary tooth) co-occurring with severe pigmentation defects. The long term goal of my research program is to identify and functionally validate the loci causing this spectrum of craniofacial abnormalities in our natural model system, Astyanax mexicanus. This R03 application, which represents a significant step towards attaining this goal has two specific aims: 1) define the genetic bases for craniofacial abnormalities and pigmentation defects in the context of a densely-populated Astyanax linkage map, and 2) identify candidate genes governing craniofacial alterations utilizing tissue-specific whole genome transcriptome profiling. The central hypothesis of this research is that multiple degenerative phenotypes present in the natural world arise through the same, or closely linked, pleiotropic loci. The proposed project will utilize high-resolution microCT X-ray imaging of a large linkage-mapping fish pedigree. This non-invasive approach facilitates co-analysis of additional traits that may be influenced by the same genetic regulators. Preliminary studies indicate a genetic basis for 12 craniofacial and dental features in Astyanax, with overlap of numerous loci involved in other non-skeletal features (e.g., vision loss, pigmentation reduction). This study seeks to lay the groundwork for future applications by accelerating discovery of candidate genes involved in this constellation of phenotypes. In sum, this project represents an innovative integration of linkage mapping, next-generation sequencing approaches, and high-resolution phenotypic analyses to determine the genetic basis for craniofacial and dental abnormalities. The proposed research is significant because it will enable subsequent definitive studies at the R01 level, and is the first step in a continuum of research designed to 1) inform the precise functional role of genes causing craniofacial defects with associated co-morbid phenotypes, and 2) clarify mechanisms governing normal craniofacial and dental development in vertebrates. PUBLIC HEALTH RELEVANCE: The proposed research will utilize a novel approach to understanding how the genetic constitution of an organism contributes to craniofacial and dental abnormalities. Using a natural model system for degenerative bone formation, this work has the potential to identify loci, shared broadly across animal species including humans, which are vulnerable to mutation. The proposed research is therefore relevant to NIDCR's mission to apply rigorous, multidisciplinary approaches to improve our understanding of the genetic basis for craniofacial and dental disease states.